5 Abstract The most heat stable enzyme in lemon and other citrus juices is pectinesterase (PE). This enzyme induces pectin destabilization, which causes cloud loss in the juice. The cloud presents the fresh-like property and therefore product satisfaction. Inactivation of PE is generally used as an indicator of the adequacy of pasteurization because it is known to be more heat resistant than the common micro-organisms. Ultrasonic treatment is one of the emerging tools that could be the alternative to thermal processing. It can enhance convective heat transfer as well as generate bubble explosion, which produce local hot spot that can cause micro-organism and enzyme destruction. However, ultrasonication (US) alone cannot inactivate the thermo-stable PE, even at long exposures. The combination of ultrasound and heat (thermosonication, TS) can slightly decrease the activity of this enzyme, which depended on time and temperature. Manothermosonication (MTS) is a method of combining ultrasonication with thermal and pressure treatment. This method can significantly decrease the activity of PE at the moderate pressure ( kpa) of temperature below 100 C. Almost complete enzyme inactivation (94% inactivation at 70 C, 300 kpa, 2 min and 96% inactivation at 80 C, 200 kpa, 5 min) occurred under the conditions mentioned. The extent of inactivation depended on ph, time of exposure, temperature, pressure and amplitude of the ultrasound. Lowering the ph of the medium increased the inactivation of the enzyme. PE activity of 0.55 unit/ml was obtained at ph 2.5, 30 C, 1 min whereas 2.5 unit/ml was obtained at ph 7.5, 30 C, 1 min. Increase in time of exposure (46% increased inactivation from 3 min to 38 min at 70 C, 65% increased inactivation from 3 min to 63 min at 80 C), temperature (for 3 min treatment time; 3.7% inactivation at 40 C, 95% inactivation at 80 C, 98% inactivation at 90 C), pressure (at 70 C, 2 min PE was 26% inactivation at 100 kpa and 35% inactivation at 300 kpa) and ultrasonic power (at 80 C, 300 kpa, PE was 83.5% inactivation at ultrasonic power of 20%, 87% inactivation at power of 50% and 91% inactivation at power of 100%) enhanced enzyme inactivation. The improvement of the inactivation can be

6 represented by pressure enhancing US (e.g. for MTS inactivation of PE, D 60(100 kpa) = 3.6 min, D 60(300 kpa) = 1.18 min), US enhancing heat and pressure (e.g. for the experiment of MTS inactivation of PE in fresh lemon juice at 75 C, 300 kpa, 60% inactivation without ultrasound, 77% inactivation at 20% ultrasonic power, 81% inactivation at 50% ultrasonic power) and temperature-pressure treatment inducing chemical reactions. The decimal reduction times of MTS inactivation of tomato were also dramatically decreased. For pectinesterase, D-value of 7.39 min at 60 C, 400 kpa, 100% ultrasonic power was obtained where D-value of 21 min was obtained at 60 C without MTS treatment. The same phenomenon was observed in polygalacturonase (D 60 = min, D 60(MTS) = 5.63 min) peroxidase (D 60 = 21 min, D 60(MTS) = 7.4 min) and polyphenoloxidase (D 60 = 14.7 min, D 60(MTS) = 8.9 min) inactivation. These inactivations depended on temperature (e.g. PE; D 60 = 12.8 min, D 80 = 1 min) and time (e.g. PE activity at 70 C, 1 min = 0.49 unit/ml, PE activity at 70 C, 5 min = 0.08 unit/ml). Further investigation should focus on the mechanisms of the combination treatment. MTS treatment was also investigated on fresh lemon juice and strawberry juice. It has been shown the great potential of this new technology since the MTS treatment could maintain properties such as cloud, colour, ph and conductivity. However, in terms of the nutrition value, ascorbic acid undergoes degradation during the treatment and storage. One needs to investigate further on the optimum treatment of MTS (e.g. oxygen removal) in order to preserve the nutritional indicators in lemon juice.

8 Acknowledgement Firstly and foremost, my sincere gratitude goes to Prof. Dr. D. Knorr for his support. He has given me an academic guidance and kept inspiring me throughout my study. He also stood by me every time I faced problems and helped me to keep perspective. I wish to thank Dr. M. N. Eshtiaghi, who has supported me not only proof-read multiple versions of all chapters of this dissertation, but also provided many suggestions to improve my presentation and clarify my arguments. My research was necessary to use several laboratory resources. Therefore, I am truly thankful to M. Bunzeit for performing some biological experiments, M. Zenker for controlling ultrasonic equipment and suggesting on my publications, and A. Angersbach for showing me some techniques of Plot-it software programme, which I have used so frequently. My thanks go to Dr. V. Heinz and all people of Food Technology Department, Technical University of Berlin for welcoming and supporting me so warmly to Berlin. I wish to thank W. Tedjo, who was willing to read my work and thus provided some very useful input. The writing of a dissertation can be a lonely and isolating experience; it is obviously not possible without personal support from numerous people. Thus, I would like to give my special thanks to the Frädrich family for giving me frequent respite and much love, and particularly to P. Euasookkul for her boundless hospitality and consistent encouragement. I wish to thank my friend, S. Muanpawong, for her substantive supports and discussions that pushed me much effort over the past years, and all of my friends and companion in Germany for all helps and supports. My enormous debt of gratitude to my family for their love, support and unlimited patience can hardly be repaid. My father is not only a sponsor of my study, but he is also a sponsor of my life and career. Finally, it was a great opportunity to study in Germany. I have had a valuable time and experience that I would never forget. Therefore, I would like to pay my tribute to German study system that provides students extensive facilities, opportunities and knowledge.

9 Table of contents Part I Introduction...1 Chapter 1 General consideration and aims...2 Part II Theory...4 Chapter 2 Ultrasonic science Sound ranges Mechanisms and effects...6 Chapter 3 Effect of ultrasound on enzyme inactivation Inactivation kinetics of enzymes Evaluation of D and z values of enzyme for thermal process calculation Order of the reaction Temperature dependence Transition state theory and pressure dependence Application of ultrasound on enzyme inactivation General information Application of ultrasound Ultrasonication Presonication Thermosonication Postsonication Manosonication Manothermosonication...15 Chapter 4 Application of ultrasound in food industry General information about ultrasound in food industry Diagnostic ultrasound Power ultrasound Acceleration of the reaction Cleaning and degassing liquids...18 i

13 Content of tables Table 4.1: Literature summary of citrus pectinesterase inactivation and ultrasonic application on enzyme inactivation...26 Table 5.1 Actual power and amplitude of the ultrasonication in discontinuous unit...34 Table 5.2 Ultrasonic equipment in the continuous process...34 Table 5.3: Actual power and amplitude of the ultrasonication in continuous unit...35 Table 5.4: Temperature and actual power during thermal treatment and thermosonication (without pressure)...37 Table 5.5: Temperature and power during the combination treatment of temperature and pressure and manothermosonication...39 Table 5.6: Mass flow and the holding time in the equipment...42 Table 6.1: Effect of ph on the PE activity (at 30 C)...52 Table 6.2: D-values of heat and thermosonication PE inactivation...54 Table 6.3: D-values and inactivation rate constant of heat treatment...56 Table 6.4: Temperature and pressure inactivation of thermoresistant PE (see Appendix A.4, A.5, A.6)...56 Table 6.5: D-values and V a of the combination treatment of temperature and pressure...57 Table 6.6: D-values and V a of manothermosonication treatment...59 Table 6.7: z-values and E a of manothermosonication treatment...60 Table 6.8: The D-value (min) of thermal treatment and the combination treatment of heat, pressure and ultrasound on PPO...62 Table 6.9: The D-value (min) of thermal treatment and the combination treatment of heat, pressure and ultrasound on POD...64 Table 6.10: The D-value (min) of thermal treatment and the combination treatment of heat, pressure and ultrasound on PE...65 Table 6.11: The D-value of thermal treatment and the combination treatment of heat, pressure and ultrasound on PG...67 A.1: Heat inactivation of pectinesterase extracted from fresh lemon at various temperature (see figure 6.1)...85 v

15 Content of figures Figure 5.1: The flow chart of the strawberry process...32 Figure 5.2: Ultrasonic horn 20 khz...33 Figure 5.3: The continuous MTS equipment and the alternative process of the combination treatment with CO Figure 5.4: The calibration curve of the holding time vs. the HPLC flow rate...37 Figure 5.5: Ultrasonication power during pressure treatment...38 Figure 5.6: Ultrasonication power during thermal treatment...38 Figure 5.7: The continuous system of MTS ( cm 3 treatment volume)...40 Figure 5.8: The calibration curve of sprectrophotometry with phenol red...44 Figure 6.1: Heat inactivation of PE at various temperatures...50 Figure 6.2: D-Value of PE inactivation at various temperatures...51 Figure 6.3: Heat and thermosonication inactivation of thermoresistant lemon PE...53 Figure 6.4: D-value of PE inactivation by heat and thermosonication...54 Figure 6.5: Thermo-stable PE inactivation at various temperatures...55 Figure 6.6: log (D) vs. temperature plot of PE heat treatment...57 Figure 6.7: Manothermosonication inactivation of thermoresistant PE...58 Figure 6.8: D-value of manothermosonication inactivation...59 Figure 6.9: The influence of temperature with and without ultrasound treatment on PPO activity...62 Figure 6.10: The influence of temperature with and without ultrasound treatment on POD activity...63 Figure 6.11: The influence of temperature with and without ultrasound treatment on PE activity...65 Figure 6.12: The influence of temperature with and without ultrasound treatment on PG activity...66 Figure 6.13: The effect of the combination treatment of ultrasound with CO 2 at 400 kpa, 60 C on PPO, POD, PE and PG activity...68 Figure 6.14: The percentage of lemon PE inactivation by manothermosonication treatment (300 kpa)...69 vii

16 Figure 6.15: The effect of different level of ultrasound on the manothermosonication inactivation of thermoresistant lemon PE...70 Figure 6.16: The untreated and treated lemon juices after 18 days (stored at 4 C)...73 Figure 6.17: Ascorbic acid of the MTS treated juice at 300 kpa...74 (immediate after treatment)...74 Figure 6.18: Ascorbic acid of the MTS treated juice at 300 kpa...75 (after 16 storage days, 4 C)...75 Figure 6.19: The percentage of strawberry PE inactivation by...76 manothermosonicaion at 300 kpa...76 Figure 6.20: The effect of different level of ultrasound on the manothermosonication inactivation of thermoresistant strawberry PE...77 B.1: Temperature profile of treated enzyme at the outlet from the combination treatment of temperature, pressure 100 kpa and sonication power 100% in the lemon PE continuous process...99 B.2: Temperature profile of treated enzyme at the outlet from the combination treatment of temperature, pressure 200 kpa and sonication power 100% in the lemon PE continuous process...99 B.3: Temperature profile of treated enzyme at the outlet from the combination treatment of temperature, pressure 300 kpa and sonication power 100% in the lemon PE continuous process B.4: Temperature profile during the combination process of ultrasound, heat and pressure B.5: The untreated lemon juice B.6: The treated lemon juice at 60 C, 300 kpa with and without ultrasonic treatment (after the treatment and 18 storage days) B.7: The treated lemon juice at 70 C, 300 kpa with and without ultrasonic treatment (after the treatment and 18 storage days) B.8: The treated lemon juice at 70 C, 300 kpa with and without ultrasonic treatment (after the treatment and 18 storage days) B.9: The treated lemon juice at 75 C, 300 kpa with and without ultrasonic treatment (after the treatment and 18 storage days) viii

21 Chapter 1 General consideration and aims Commercial lime and lemon juices are among the world most important citrus products. For example, these juices are used as a common ingredient in most of the traditional Asian cooking. Moreover, they are necessary for many global food productions, such as lemonade drink, marmalade, jams, candies, jellies, desserts, pharmaceutical products, and medicines. Lemon juice, itself, is a colloidal suspension of cellular and polymer particles. This cloudy appearance is an important property of the juices since it gives the natural appeal of the fresh juices. Colloidal stability is maintained by pectin molecules through a complex and not well understood mechanism. Cloud loss of citrus juices is an intensively studied problem in food technology. It is due to the action of endogenous pectinesterase (PE) on pectin substance. PE catalyzes the de-esterification of pectin molecules. De-esterified pectin molecules are able to interact through calcium bridges, leading to cloud loss and phase separation in single-strength lemon juices and gelation in their concentrates. Stabilization of cloud in citrus juices requires the inactivation or inhibition of PE (Vercet, et al., 1999). Several strategies have been used to inhibit or inactivate PE avoiding the negative effects of intensive heat treatments. Inhibition of PE by polyphenols (Hall, 1966; Pilnik and Voragen, 1991), inhibition by specific proteic PE inhibitors (Castaldo et al., 1991), or inhibition by the oligogalacturonides produced by the action of added polygalacturonase or pectinylase (Baker and Bruemmer, 1972; Krop and Pilnik, 1974; Termote et al., 1977) have been suggested as alternatives to the heat treatments. Other strategies rely on PE 2

22 CHAPTER 1 GENERAL CONSIDERATION AND AIMS inactivation by nonthermal treatments such as high pressure (Irwe and Olson, 1994; Donsi et al., 1996; Cano et al., 1997; Knorr, 1998), low ph values (Owusu-yaw et al., 1988), or supercritical carbon dioxide (Arreola et al., 1991; Balaban et al., 1991; Ishikawa at al., 1996). Another possible alternative is ultrasound in combination with heat and pressure (Manothermosonication; MTS). MTS is an emerging technology that efficiently combines the inactivating effect of heat and ultrasonic waves (Burgos, 1998). MTS has been proved to be an efficient tool to inactivate some other enzymes, such as lipoxygenase, peroxidase, and proteases and lipases from psychrophic bacteria (Lopez at al., 1994; Sala at al., 1995; Vercet at al., 1997). Most results reported in the scientific literature, in fact, relate deactivating and destructive action of ultrasound only to its frequency and fail to provide information about the dependence of the treatment efficiency on the actual power and power density of ultrasound. In addition, no definite experimental evidence has been reported on the efficiency of ultrasound in batch or in continuous applications. The aims of the study were to investigate the effect of ultrasound on the inactivation of lemon pectinesterase and juice quality. One further aim was the evaluation of the kinetic parameters of the MTS effect on lemon and tomato pectinesterase. Finally, the potential of the MTS in fruit and vegetable (lemon, strawberry, tomato) juice industry was determined. 3

23 Part II Theory

24 Chapter 2 Ultrasonic science 2.1 Sound ranges The range of human hearing is from about 16 Hz to 18 khz. Ultrasound is the name given to sound waves having frequencies higher than those to which the human ear can respond (i.e. > 18 khz). The upper limit of ultrasound frequency is one, which is not sharply defined but is usually taken to be 5 MHz in gases and 500 MHz in liquids and solids. The use of ultrasound within this large frequency range may be divided broadly into two areas. The first area involves low amplitude (higher frequency) propagation, which is concerned with the effect of the medium on the wave and is commonly referred to as low power or high frequency ultrasound. Typically, low amplitude waves are used to measure the velocity and absorption coefficient of the wave in a medium in the 2 to 10 MHz range. It is used in medical scanning, chemical analysis and the study of relaxation phenomena. The second area involves high energy (low frequency) waves known as power ultrasound between 20 and 100 khz which is used for cleaning, plastic welding and, more recently, to effect chemical reactivity. Ultrasonic waves are generated by mechanical vibrations of frequencies higher than 18 khz. When these waves propagate into liquid media, alternating compression and expansion cycles are produced. During the expansion cycle, high intensity ultrasonic waves make small bubbles grow in liquid. When they attain a volume at which they can no longer 5

25 CHAPTER 2 ULTRASONIC SCIENCE absorb enough energy, they implode violently. This phenomenon is known as cavitation. During implosion, very high temperatures (approximately 5000 K) and pressures (estimated at kpa) are reached inside these bubbles (Sala et al., 1998). 2.2 Mechanisms and effects High-intensity acoustic radiation causes various changes as is propagates through a medium. These changes can be explained by several mechanisms, but not all mechanisms involved are known or well understood. Most of the reported effects and proposed mechanisms can be summarized as follows: Heating: As a result of specific absorption of acoustic energy by membranes and biomaterials, particularly at their interfaces, a selective temperature increase may take place (Floros and Liang, 1994). This heating effect was assumed to be responsible for the significant increase in diffusion of sodium ions through living frog skin under ultrasound (Lehmann and Krusen, 1954). The increase in permeability of the living membrane was so large that its selectivity was completely lost. Later theoretical and experimental results do not support the early assumptions. Floros and Liang (1994) emphasized about the heat balance equation to calculate loss of ultrasonic energy as it propagates through a medium. They mentioned that the temperature change due to absorption at a solid wall, under given conditions, was 0.1 C for water and about 1 C for air. These results were verified experimentally. Other investigators claim that localized temperature increase of up to 5000 K can be expected for a few nanoseconds in a sound field (Suslick et al., 1985). Cavitation: Acoustic cavitation is the formation, growth, and violent collapse of small bubbles or voids in liquids as a result of pressure fluctuation (Suslick, 1988). In general, cavitation in liquids may cause fast and complete degassing; initiate various reactions by generating free chemical ions (radicals); accelerate chemical reactions by facilitating the mixing of reactants; enhance polymerization/ depolymerization reactions by temporarily dispersing aggregates or by permanently breaking chemical bonds in polymeric chains; increase emulsification rates; improve diffusion rates; produce highly concentrated 6

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